Tochingyuet (Talk | contribs) |
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p{ | p{ | ||
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font-family: roboto; | font-family: roboto; | ||
} | } | ||
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+ | section { | ||
+ | font-family: roboto; | ||
+ | margin:auto; | ||
+ | position: relative; | ||
+ | width: 85%; | ||
+ | height: auto; | ||
+ | } | ||
</style> | </style> | ||
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</div> | </div> | ||
<div class="column full_size"> | <div class="column full_size"> | ||
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<br><br> | <br><br> | ||
− | <p style="font-family: roboto;font-size:150%;">Below | + | <p style="font-family: roboto;font-size:150%;">Below are the protocols that we used in our project. Click the boxes for more detail! </p> |
<br> | <br> | ||
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<li>Start the cycle and wait till it is finished. </li> | <li>Start the cycle and wait till it is finished. </li> | ||
</ol> | </ol> | ||
+ | <a href="https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530">Click here for the manufacturer's maual</a> | ||
</p> | </p> | ||
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<li>Incubate overnight at 37°C with plates upside down.</li> | <li>Incubate overnight at 37°C with plates upside down.</li> | ||
</ol> | </ol> | ||
+ | <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846332/">Click here for the manufacturer's maual</a> | ||
</div> | </div> | ||
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<li>Add 30 μl of distilled water to elute DNA.</li> | <li>Add 30 μl of distilled water to elute DNA.</li> | ||
</ol> | </ol> | ||
+ | <a href=" http://eng.intronbio.com/PROTO-PDF/mega-spin.pdf">Click here for the manufacturer's maual</a> | ||
</div> | </div> | ||
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<div class="panel-body"> | <div class="panel-body"> | ||
<ol> | <ol> | ||
− | <li>Prepare the | + | <li>Prepare the reagents as follow:</li> |
<table> | <table> | ||
<tr> | <tr> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>DNA </td> | + | <td>DNA template</td> |
− | <td>0. | + | <td>0.5 μg</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>10X Buffer</td> |
− | <td> | + | <td>2.5 μl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>Enzyme</td> |
<td>2 μl</td> | <td>2 μl</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>Nuclease-Free Water to final colume of:</td> |
− | + | <td> 25 μl</td> | |
− | + | ||
− | + | ||
− | + | ||
− | <td> | + | |
</tr> | </tr> | ||
</table> | </table> | ||
− | <li> | + | <li>Pipette the solution up and down to ensure all reagents are mixed well.</li> |
− | <li> | + | <li>Place the reaction mixture at 37 oC incubation or dry bath for 2-4 hours. </li> |
+ | <li>Purify the DNA by PCR purification kit/gel extraction kit for downstream process. </li> | ||
</ol> | </ol> | ||
+ | <a href=" https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012178_XbaI_10_UuL_5X1500U_UG.pdf">Click here for the manufacturer's maual</a> | ||
</p> | </p> | ||
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<div class="panel-body"> | <div class="panel-body"> | ||
<ol> | <ol> | ||
− | <li>Prepare the | + | <li>Prepare the reaction mixture as follow:</li> |
<table> | <table> | ||
<tr> | <tr> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>DNA | + | <td>DNA </td> |
− | <td>0. | + | <td>0.2M KCl/HCl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>DNA template</td> |
− | <td> | + | <td>10-200 ng</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>10XT4 DNA Ligase Reaction Buffer</td> |
<td>2 μl</td> | <td>2 μl</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>T4 DNA Ligase</td> |
− | <td> | + | <td>1 μl</td> |
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Water</td> | ||
+ | <td>Top up to 20 μl</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | <li> | + | <li>Allow the ligation to take place a 22-25°C for 10 minutes.</li> |
− | <li> | + | <li>Send 5 μl of the ligated sample should be used for agarose gel electrophoresis to confirm whether ligation has occurred (Optional)</li> |
− | + | ||
</ol> | </ol> | ||
+ | |||
+ | <a href=" https://www.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202">Click here for the manufacturer's maual</a> | ||
</p> | </p> | ||
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<li>Test the transformation efficiency of competent cells with antibiotics resistance plamids at different concentrations.</li> | <li>Test the transformation efficiency of competent cells with antibiotics resistance plamids at different concentrations.</li> | ||
</ol> | </ol> | ||
− | + | <a href=" https://www.neb.com/protocols/2012/06/21/making-your-own-chemically-competent-cells">Click here for the manufacturer's maual</a> | |
</p> | </p> | ||
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<li>Incubate overnight at 37°C with plates upside down.</li> | <li>Incubate overnight at 37°C with plates upside down.</li> | ||
</ol> | </ol> | ||
+ | <a href=" https://www.neb.com/protocols/2012/05/21/transformation-protocol">Click here for the manufacturer's maual</a> | ||
</div> | </div> | ||
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<li>Centrifuge at 13,000 rpm for 1 min.</li> | <li>Centrifuge at 13,000 rpm for 1 min.</li> | ||
</ol> | </ol> | ||
− | + | <a href=" http://eng.intronbio.com/PROTO-PDF/DNA-spin.pdf">Click here for the manufacturer's maual</a> | |
</p> | </p> | ||
</div> | </div> | ||
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</ul> | </ul> | ||
</ol> | </ol> | ||
− | + | <a href=" https://www.thermofisher.com/hk/en/home/life-science/cell-culture/microbiological-culture/bacterial-growth-media/lb-broth-and-lb-agar.html">Click here for the manufacturer's maual</a> | |
</div> | </div> | ||
</div> | </div> | ||
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<li>Analyze the results of the reaction. </li> | <li>Analyze the results of the reaction. </li> | ||
</ol> | </ol> | ||
− | + | <a href=" https://www.promega.com/-/media/files/resources/protocols/technical-manuals/0/s30-t7-high-yield-protein-expression-system-protocol.pdf">Click here for the manufacturer's maual</a> | |
</p> | </p> | ||
</div> | </div> | ||
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− | </ | + | </body> |
+ | </section> | ||
− | |||
</html> | </html> | ||
+ | {{Template:Hong_Kong-CUHK_footer}} |
Latest revision as of 14:44, 30 October 2017